Fig 1.
Volumetric analysis of GL261 gliomas through quantification of T2 and T1 gadolinium enhanced MRI in vivo.
(A,B) Volumetric analysis and 3D rendering of a representative live mouse bearing a GL261 glioma as visible by T2 weighted MRI using Analyze 11.0 software. (C) Quantification of mean tumor volumes demonstrates larger values from T2 weighted MRI than T1 gadolinium-enhanced MRI for both GL261 (N = 3 mice) and GL261-Quad (N = 4 mice) gliomas 4 weeks post tumor injection compared to PBS sham controls (N = 2 mice). (D) T2 weighted and T1 gadolinium-enhanced MRIs with corresponding bioluminescence images of representative small and large GL261-Quad gliomas. (E) Tumor volumes obtained from MRI plotted against bioluminescence intensity demonstrate a strong correlation for both T2 weighted (R2 = 0.9045) and T1 gadolinium-enhanced (R2 = 0.9296) imaging (N = 15 mice/group). (F,G) Representative H&E stained sections at 4X and 10X magnification of cortical brain tissue from mice bearing GL261 or GL261-Quad gliomas demonstrate similarities in histopathologic features of the two models.
Fig 2.
GL261-Quad gliomas elicit an endogenous Kb:OVA257–264 restricted CD8+ T cell response.
Immune cells from whole brain tissue of glioma-bearing mice were isolated and gated based on CD45 expression. CD45Hi cells were further analyzed for CD8, CD4, CD11b, GR-1 and Kb:OVA257–264 tetramer positivity. (A) The inflammatory profile of a representative GL261-bearing mouse. (B) Relative percentages of immune cell subsets in GL261 glioma-bearing mice confirm defined populations of CD8+, CD4+, and CD11b+,GR-1+ cells (N = 3 mice). (C) The inflammatory profile of a representative GL261-Quad glioma-bearing mouse. (D) Kb:OVA257–264 tetramer staining shows the presence of endogenous tumor antigen-restricted CD8+ T cell responses in the absence of treatment in mice bearing GL261-Quad gliomas (N = 5 mice).
Fig 3.
Vaccination with TMEV Xho1-OVA8 enhances tumor antigen-restricted CD8+ T cell responses in the CNS, delays tumor progression, and extends survival in mice bearing GL261-Quad gliomas.
Two weeks post-GL261-Quad tumor introduction, mice were treated with control or recombinant picornavirus. (A,B) At five weeks, brain infiltrating lymphocytes were isolated, gated based on CD45 expression, and analyzed for CD8 and Kb:OVA257–264 tetramer positivity. (A) Representative FACS plots and (B) mean percent of Kb:OVA257–264+ cells per CD8+ cells demonstrate a significant increase in tumor antigen-restricted CD8+ T cells infiltrating the brain following intracranial (N = 9) or intraperitoneal (N = 10 mice) TMEV Xho1-OVA8 vaccine administration compared to TMEV-wt. Percent of Kb:OVA257–264+ / CD8+ calculated as Q2-2/(Q2-2+Q4-2)*100. (C,E) Mean bioluminescence intensity (p/sec) of GL261-Quad glioma-bearing mice treated with TMEV-wt or recombinant TMEV Xho1-OVA8 picornavirus. Mice receiving (C) intracranial (N = 15 mice) or (E) intraperitoneal (N = 13 mice) TMEV Xho1-OVA8 treatment displayed significantly delayed progression of established gliomas compared to TMEV-wt treated controls. (D,F) Delayed tumor progression is accompanied by a significant increase in survival for mice treated with TMEV Xho1-OVA8 via (D) intracranial or (F) intraperitoneal administration. Arrow denotes time of vaccine administration. (G) RT-PCR analysis of RNA isolated from brains of glioma-bearing mice demonstrates a significant reduction in transgene expression following treatment with TMEV Xho1-OVA8 compared to TMEV-wt treated controls. Error bars indicate SEM. * denotes p<0.05, ** denotes p<0.01, ***denotes p<0.001.
Fig 4.
Loss of CD8+ T cell cytotoxicity abrogates the effects of picornavirus vaccination against GL261-Quad gliomas.
(A) Mean bioluminescence intensity (p/sec) of wild type C57BL/6 or perforin deficient (Prf-/-) GL261 glioma-bearing mice demonstrates no difference in the rate of tumor progression in the absence of treatment (N = 18 mice/group). (B) Mean BLI (p/sec) and (C) survival curve of C57BL/6 (N = 10 mice/group) or Prf-/- (N = 7 mice) GL261-Quad glioma-bearing mice following picornavirus vaccination demonstrates a complete loss of vaccine efficacy in the absence of functional cytotoxic CD8+ T cell activity. Error bars indicate SEM. * denotes p<0.05, ** denotes p<0.01, ***denotes p<0.001. Arrow denotes time of vaccine administration.